Treatment of sickle cell disease

ABSTRACT

The present invention includes embodiments for treatment and/or prevention of sickle cell disease that employ Hydroxyfasudil or Isocoronarin D alone or either in conjunction with each other or an inducer of HbF production. The compounds may act synergistically, and the compounds employed circumvent the side effects seen with Hydroxyurea.

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/534,449, filed Sep. 14, 2011, which application isincorporated by reference herein in its entirety.

TECHNICAL FIELD

The field of the present invention includes at least biology, cellbiology, and medicine. In particular aspects, the field of the presentinvention includes treatment and/or prevention of a blood disorder, suchas sickle cell disease.

BACKGROUND OF THE INVENTION

Sickle cell disease (SCD) is the most common life-threatening monogenicdisorder in the world with statistics indicating that approximately 80%(230,000) of children affected globally are born in sub-Saharan Africa(Modell and Darlison 2008). SCD is a severe hemoglobinopathy thatproduces multisystem complications due to the expression of abnormalsickle hemoglobin (HbS). The most common type of SCD is sickle cellanemia (SCA) (also referred to as HbSS or SS disease or hemoglobin S) inwhich there is homozygosity for the mutation that causes HbS. The morerare types of SCD in which there is heterozygosity (one copy of themutation that causes HbS and one copy for another abnormal hemoglobinallele) for the mutation include sickle-hemoglobin C (HbSC), sickle β⁺thalassemia (HbS/β⁺) and sickle β⁰ thalassemia (HbS/β⁰).

Sickle cell disease (SCD) can arise from a single point mutation thatcauses erythrocyte deformation or sickle-shaped erythrocytes (Ingram1957). Sickled-shaped erythrocytes are associated with clinicalmanifestations of SCD, such as anemia, recurrent painful vaso-occlusiveepisodes, infections, acute chest syndrome, pulmonary hypertension,stroke, priapism, osteonecrosis, renal insufficiency, leg ulcers,retinopathies, and cardiac disease (Frenette and Atweh 2007, Steinberg2008).

Pathophysiology

Sickle-Shaped Erythrocytes

SCD arises from a single point mutation (GAG>GTG) in codon 6 of the HBBglobin gene. This point mutation (GAG>GTG) results in the polarhydrophilic molecule, glutamic acid being substituted with the non-polarhydrophobic molecule, valine (β⁶ Glu→Val) (Ingram 1957). Thedeoxygenated venous circulation causes the hydrophobic valine residue toassociate with hydrophobic regions of adjacent molecules (Mozzarelli, etal 1987). This process of self-assembly (polymerization) generates thesickled hemoglobin molecule (HbS) that damages the membrane andcytoskeleton of the erythrocyte. The HbS repetitively enter intosickling and unsickling cycles incrementally increasing the damage tothe erythrocyte membrane (Ischemia-reperfusion (IR) injury) resulting inirreversibly sickle-shaped erythrocytes (Barabino, et al 2010).

C-reactive protein (CRP) (Nath, et al 2005) and the markers of oxidativestress, such as xanthine oxidase (XO), salicylate hydroxylation andexpired ethane, are significantly increased following IR injury (Huang,et al 2007, Osarogiagbon, et al 2000). The activation of XO and reducedTetrahydrobiopterin (BH₄) levels in endothelial cells leads to increasedreactive oxygen species (ROS) formation, which in turn leads toendothelial nitric oxide synthase (eNOS) uncoupling and furtherproduction of superoxide (Thomas, et al 2010). ROS causes endothelialcell membrane lipid peroxidation, inactivation of nitric oxide (NO),activation of nuclear factor of kappa light polypeptide gene enhancer inB-cells 1 (NF-κB) and Src/MAP kinase signaling, and induces productionof tissue factor (TF), interleukin 8 (IL-8), and surface adhesionmolecule expression (Belcher, et al 2006, Radi, et al 1991, Thomas, etal 2010). Thus, the ensuing oxidative stress contributes to hemolysis,inactivation of NO, and erythrocyte, leukocyte and platelet adhesiveproperties (Kaul, et al 2004, Sultana, et al 1998, Vilas-Boas, et al2010).

The Sickled-shaped erythrocytes together with endothelial cells,activated leukocytes, platelets and plasma proteins participate in themultistep vaso-occlusion process (Frenette 2002).

TABLE 1 Ischemia-reperfusion (IR) injury-related molecules inindividuals with SCD Expression IR injury-related molecules status inSCD References Xanthine oxidase (XO) Up (Osarogiagbon, et al 2000)Tetrahydrobiopterin (BH₄) Down (Thomas, et al 2010) C-reactive protein(CRP) Up (Nath, et al 2005) Reactive Oxygen Species Up (Huang, et al2007) (ROS)

Endothelial Cells

Vascular homeostasis is harmonized by the endothelial cellvasoregulators regulating blood flow, growth of vascular smooth musclecells and local inflammation (Huang, et al 2006). Endothelial cells arethe primary producers of the major vasodilator, nitric oxide (NO) andprostacyclin, as well as vasoconstrictors such as endothelin,angiotensin II and prostaglandins (Galley and Webster 2004). Theseendothelial cell vasoregulators are characteristically imbalanced inindividuals with SCD, resulting in endothelial dysfunction thatcontributes to vaso-occlusion process.

SCD is characterized by a reduced bioavailability of NO, due to1/cell-free plasma hemoglobin and increased arginase activityencouraging hemolysis-related scavenging of NO (Reiter and Gladwin2003), and 2/eNOS function being diminished due to a decrease in thenitric oxide synthase (NOS) substrate, arginine's ability to dimerizethereby contributing to reduced NO synthesis (Lin, et al 2011). Thereduced NO decreases the NO-dependent vaso-dilation, contributing to anincrease in vasoconstrictors such as endothelin-1 (ET-1) (Ergul, et al2004, Werdehoff, et al 1998) and endothelin-3 (ET-3) (Makis, et al2004). It has been demonstrated that Endothelin-3 induces endothelialcell interleukin 6 (IL-6) expression thereby mediating inflammation.Thus, IL-6 is also characteristically found to be increased in SCDpatients (Makis, et al 2004).

TABLE 2 Endothelial cell vasoregulators that is imbalanced inindividuals with SCD Endothelial Expression Cell Type of status inVasoregulators Vasoregulator SCD References Nitric Oxide vasodilatordown (Eberhardt, et al 2003) Endothelin-1 vasoconstrictor up (Werdehoff,et al 1998) Endothelin-3 vasoconstrictor up (Makis, et al 2004)

Activators of endothelial cells include NFκB (Belcher, et al 2005),hypoxia-inducible factor-1 (HIF-1) (Kim, et al 2006), ET-1 (Phelan, etal 1995) and TF (Solovey, et al 1998). Interestingly, it has beendemonstrated that eNOS modulates the expression of TF by down-regulatesit, however, eNOS is characteristically down-regulated in SCD patients(Solovey, et al 2010). Once activated, the endothelium producescytokines such as tumour necrosis factor alpha (TNFα) (Lanaro, et al2009) and interleukin 1 beta (IL-1β) (Croizat 1994, Wanderer 2009),chemokines and/or inflammatory molecules such as granulocytemacrophage-colony stimulating factor (GM-CSF) (Conran, et al 2007), IL-8(Lanaro, et al 2009), IL-6 (Croizat 1994), interleukin 3 (IL-3) (Croizat1994), interleukin 4 (IL-4) (Musa, et al 2010), and platelet activatingfactor (PAF) (Oh, et al 1997), and adhesion molecules such as vascularcell adhesion molecule 1 (VCAM-1), intercellular cell adhesion molecule1 (ICAM-1), selectin E (SELE) and selectin P (SELP) (Chiu, et al 2004,Solovey, et al 1997). Rajan et al. demonstrated that NFκB is requiredfor TNFα-induced expression of VCAM-1, ICAM-1 and SELE in endothelialcells (Rajan, et al 2008). The production of these inflammatorymediators and cell adhesion molecules is perpetuated by TNFα and IL-1βbeing potent activators of the endothelium (Segers, et al 2006). Hemeoxygenase-1 (HO-1) (Belcher, et al 2006) and interleukin 10 (IL-10)(Musa, et al 2010) are characteristically found to be increased in SCDpatients in an attempt to counteract the induced inflammation. HO-1breaks down heme released during hemolysis thereby limiting oxidativestress and inflammation (Otterbein, et al 2003), whilst IL-10 limits theproduction of the pro-inflammatory cytokines (Lanaro, et al 2009,Taylor, et al 2001).

TABLE 3 Endothelial cell activation-related molecules in individualswith SCD Ex- pression status in Molecules Types of Molecules SCDReferences NFκB Activator of endothelial up (Belcher, et al 2005) cellsET-1 Activator of endothelial up (Phelan, et al 1995) cells HIF-1Activator of endothelial up (Kim, et al 2006) cells TF Activator ofendothelial up (Solovey, et al 1998) cells eNOS Inhibitor of endothelialdown (Solovey, et al 2010) cell activation TNFα cytokine/inflammatory up(Lanaro, et al 2009) IL-1β cytokine/inflammatory up (Wanderer 2009) IL-8chemokine/inflammatory up (Lanaro, et al 2009) IL-4 chemokine up (Musa,et al 2010) PAF chemokine up (Oh, et al 1997) IL-6chemokine/inflammatory up (Croizat 1994) GM-CSF inflammatory up (Conran,et al 2007) IL-3 inflammatory up (Croizat 1994) VCAM-1 adhesion up(Solovey, et al 1997) ICAM-1 adhesion up (Solovey, et al 1997) SELPadhesion up (Solovey, et al 1997) SELE adhesion up (Solovey, et al 1997)

Leukocytes

The sickled erythrocytes stimulates leukocyte recruitment: ensuing theinflammatory stimulus, leukocytes are recruited to the activatedendothelium of the venous circulation where it forms adhesiveinteractions with the activated endothelium and sickled erythrocytes,leading to a reduced blood flow and eventually vaso-occlusion (Turhan,et al 2002).

SCD patients characteristically have increased levels of activatedleukocytes. It has been demonstrated that SCD-related leukocytes haveincreased expression and activity of the integrin, beta 2 (complementcomponent 3 receptor 3 and 4 subunit) (ITGB2) LFA-1, integrin, alpha M(complement component 3 receptor 3 subunit) (ITGAM) and integrin, beta 1

(fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2,MSK12) (ITGB1) in the presence of the inflammatory stimulus, IL-8(Canalli, et al 2011, Lomakina and Waugh 2010). These SCD leukocyteintegrins have been demonstrated to ligate with endothelial ICAM-1(Canalli, et al 2011) and fibronectin 1 (FN1) (Miguel, et al 2011). Theleukocyte recruitment process is enhanced by the overexpression SELP andSELE in endothelial cells as endothelial cells lacking SELP and SELEhave demonstrated reduced leukocyte recruitment and diminishedvaso-occlusion (Turhan, et al 2002). Additionally, the lack of SELE hasalso been demonstrated to reduce the adhesion of sickled erythrocytes toleukocytes (Hidalgo, et al 2009).

TABLE 4 Leukocyte activation-related molecules in individuals with SCDExpression status Molecules Types of Molecules in SCD References ITGB2leukocyte integrin up (Canalli, et al 2011) ITGAM leukocyte integrin up(Canalli, et al 2011) ITGB1 leukocyte integrin up (Canalli, et al 2011)

Platelets

SCD plateles show increased surface expressions of SELP, activatedα_(IIb)β₃ (GPIIbIIIa) (Devi, et al 2010) and higher concentrations ofthe platelet activation markers, platelet factor 4 (PF-4) and βthromboglobulin (TGB) (Westwick, et al 1983, Yoong, et al 2003).Activated platelets have been shown to release endothelium activators,such as soluble CD40 ligand (sCD40L) (Lee, et al 2006), PF-4 (Westwick,et al 1983) and IL-1β (Wun, et al 2002). In healthy individuals,platelet adhesion is inhibited by the antithrombotic factor, NO whilstSCD platelet adhesion is stimulated by the activated endotheliumreleasing ADP, TF, von Willebrand factor (vWF) and the expression ofplatelet-binding adhesion molecules such as glycoprotein Ib (platelet),beta polypeptide (GPIb), selectin P ligand (SELPLG), vitronectinreceptor (VTNR) and ICAM-1 (van Gils, et al 2009). SCD platelets havebeen demonstrated to have increased adhesion to the αIIbβ3 plateletintegrin ligand, fibrinogen that in turn favors the adhesion ofplatelets to endothelium protein FN1 (Chada, et al 2006). Moreover,platelets and sickled erythrocytes have been demonstrated to aggregatevia the formation of thrombospondin bridges thereby contributing tovaso-occlusion (Wun, et al 1999).

TABLE 5 Platelet activation-related molecules in individuals with SCDExpression status Molecules Type of molecule in SCD References α_(IIb)β₃platelet activation up (Devi, et al 2010) PF-4 platelet activation/ up(Westwick, et al 1983) endothelium activators TGB platelet activation up(Yoong, et al 2003) sCD40L endothelium activators up (Lee, et al 2006)IL-1β endothelium activators up (Wun, et al 2002) GPIb adhesion up (vanGils, et al 2009) PSGL-1 adhesion up (van Gils, et al 2009) VTNRadhesion up (van Gils, et al 2009) ICAM-1 adhesion up (van Gils, et al2009) fibrinogen platelet integrin ligand up (Chada, et al 2006)

Hydroxyurea

Hydroxyurea (HU) is a FDA approved drug that is the only currenttreatment proven to modify the disease process of SCD (Brawley, et al2008). HU positively counteracts the pathophysiology of SCD byincreasing the production of fetal hemoglobin (HbF)-containingerythrocytes via stimulation of the NO-cyclic guanosine monophosphate(cGMP) signaling pathway (Cokic, et al 2003) and indirectly alteringgene expression and proteins associated with the pathophysiology of SCD.The increased concentration of HbF-containing erythrocytes dilutes theconcentration of sickled erythrocytes, thereby sequentially triggeringdecreased hemolysis (Olnes, et al 2009), increased NO bioavailability(Conran, et al 2004) and decreased endothelium activation (Haynes, et al2008), which likely accounts probably accounts for the beneficialeffects of HU treatment in SCD patients. However, HU has beendemonstrated to reduce leukocyte counts in patients on therapy(Charache, et al 1996). Although HU improved clinical symptoms byreducing pain and vaso-occlusive crises, acute chest syndrome,transfusion requirements, and hospitalization, SCD patients treated withHU have demonstrated side effect such as inducing DNA damage(Friedrisch, et al 2008), reducing sperm counts (Grigg 2007) andproducing iron nitrosyl Hb (Lockamy, et al 2003). There is a need in theart for improved SCD therapy that lacks one or more side effects of HU.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions associatedwith treatment and/or prevention of one or more blood disorders.Although in particular embodiments the blood disorder is SCD, inspecific embodiments, one or more other blood disorders may be treatedwith the present invention: a bleeding disorder (including clottingdisorders, hypercoagulability, hemophilia, or von Willebrand disease,for example), platelet disorder (essential or primary thrombocythemia orthrombocytopenia, for example), and/or hemophilia or anemia may betreated, for example. In particular embodiments of the invention, thereare methods and compositions for treatment and/or prevention of sicklecell disease (which may be referred to as sickle-cell anaemia (oranemia; SCA) or drepanocytosis). The disease may be present in a male orfemale mammal. Mammalian and/or non-human mammals may be used as sicklecell models, in certain cases. The individual treated with methodsand/or compositions of the invention may be experiencing vaso-occlusivecrisis and/or acute chest crisis, in specific cases. In specificembodiments, the individual may be experiencing or may experiencenegative side effects of a drug, such as a drug that directly orindirectly results in increased coagulation and/or increasedinflammation; in specific embodiments, the drug is HU.

Currently, Hydroxyurea (HU) is the only FDA approved drug capable ofmodifying the sickle cell disease (SCD) processes. However, due to sideeffects of HU there is a general interest for a less toxic drug for SCD.The inventors identified a drug (Hydroxyfasudil (also known as Fasudil))for the treatment of SCD that is a more suitable treatment for SCD thanthe currently administered HU therapy. Hydroxyfasudil was identified bythe inventors using an automated literature mining system that was thencomplemented by additional hand curation.

Hydroxyfasudil, a recognized Rho-kinase inhibitor, has been demonstratedto attenuate pulmonary hypertension secondary to left ventriculardysfunction that is characterized by increases in mean pulmonaryarterial pressure, pulmonary arteriolar medial thickness, active RhoA,Rhokinase II, Rho-kinase activity, endothelial nitric oxide synthase(eNOS) and endothelin-1 (ET-1) concomitant with decreased levels in NOand cGMP in the lung. As mentioned before, the effectiveness of HU forthe treatment of SCD is a consequence of its ability to produce HbF viastimulation of the NO-cyclic guanosine monophosphate (cGMP) signalingpathway (Cokic, et al 2003). Hydroxyfasudil induces increased levels ofeNOS, NO and cGMP and in specific embodiments of the invention,Hydroxyfasudil produces HbF via stimulation of the NO-cyclic guanosinemonophosphate (cGMP) signaling pathway as well (Dai, et al 2011).Additionally, IL-6 has been demonstrated to play an important role inglobin gene silencing (Ferry, et al 1997), even though increased IL-6levels have been associated with the pathophysiology of SCD.Hydroxyfasudil, on the other hand, has demonstrated the ability toreduce IL-6 levels after hypoxia/reoxygenation (H/R) injury in braintissue (Ding, et al 2010), indicating that at least in some embodimentsthat it has ability to produce HbF.

HU has also been shown to change the clinical symptoms of SCD bydecreasing the levels of vasoconstrictors such as ET-1 (Lapoumeroulie,et al 2005) and ET-3 (Makis, et al 2004), and by affecting the degree ofadherence of sickled erythrocytes and leukocytes through decreasing thelevels of endothelial adhesion molecules such as sVCAM-1, sICAM-1, sSELEand sSELP (Conran, et al 2004, Kato, et al 2005, Saleh, et al 1999).Hydroxyfasudil has been shown to suppress I/R injury-induced generationof ROS (Shiotani, et al 2004) and at least the levels ofvasoconstrictor, ET-1 (Dai, et al 2011); in specific embodiments it alsosuppresses the level of vasoconstrictor, ET-3. Hydroxyfasudil has alsobeen shown to reduce ICAM-1 expression in diabetes-induced microvasculardamage (Arita, et al 2009), as well as reduce ICAM-1, SELE and SELP,thereby diminishing leukocyte-endothelial adhesion in colonic I/R injury(Santen, et al 2010). HU therapy has also been shown to reduceerythrocyte-endothelial adhesion by reducing the gene and proteinexpression of adhesion molecules such as VLA-4 and CD36 on the surfaceof erythrocytes (Gambero, et al 2007). However, Hydroxyfasudil has beenshown to reduce HIF-la (Takata, et al 2008), TF (Satoh, et al 2010) andNFκB (He, et al 2008). Additionally, HU therapy decreased levels ofinflammatory mediators such as GM-CSF (Conran, et al 2007) and TNF-α,and it has been experimentally shown that HU therapy exert no effect onthe levels of IL-8 (Lanaro, et al 2009). However, HU has been shown toincrease levels of anti-inflammatory mediator IL-10 (Lanaro, et al2009), thereby attempting to reduce the inflammatory response but hasnot been effective. Hydroxyfasudil has been shown to decreasepro-inflammatory mediators such as TNF-α (He, et al 2008), IL-1β (He, etal 2008) and increase the anti-inflammatory mediator IL-10 (Ding, et al2010).

In certain embodiments of the invention, a compound that induces HbFproduction is employed alone or with one or more other drugs (some ofwhich may or may not induce HbF production) for the treatment of SCD.The individual may be known to have SCD or is suspected of or at riskfor having SCD. The individual may have a family history of SCD. Theindividual may be known to be a carrier of a mutation that causes SCD orbe known to have one or more biological parents that have a mutationthat causes SCD. The individual may experience one or more symptoms ofSCD, including, for example, fatigue, anemia, pain crises, dactylitis(swelling and inflammation of the hands and/or feet), arthritis,bacterial infections, splenic sequestration, liver congestion, legulcers, and so forth.

In embodiments of the invention, Hydroxyfasudil has the advantage overHU that Hydroxyfasudil has the ability to better inhibitpro-inflammatory mediators related to the degree of toxicity. Inparticular embodiments of the invention, Hydroxyfasudil is employedalone or with one or more other drugs as an alternative drug to reduceat least one symptom of SCD. In specific embodiments, Hydroxyfasudil isemployed with HU for the treatment of SCD. In embodiments whereinHydroxyfasudil is utilized with at least one other compound, those twoor more other compounds may work additively or synergistically withHydroxyfasudil.

Treatment with methods and/or compositions of the invention may begin inthe first year of life. Treatment may begin for infants or youngerchildren, for example those suffering with fever, abdominal pain,bacterial infections (including pneumococcal bacterial infections),painful swellings of the hands and feet (dactylitis), and/or splenicsequestration. Treatment may begin for adolescents and young adults,including, for example, those that develop leg ulcers, aseptic necrosis,and/or eye damage. Treatment may begin in an adult, for example onehaving intermittent pain episodes due to injury of bone, muscle, and/orinternal organs.

The treatment regimen of the invention may include one or more doses forthe individual. In some embodiments, dosage levels include a maximumdose of 700 mg/kg and a minimum dosage of 0.5 mg/kg. Dosage levelsinclude 0.5, 1, 10, 20, 25, 30, 40, 50, 60, 75, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 275, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 375,380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 475, 480, 490, 500,510, 520, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 630,640, 650, 660, 670, 675, 680, 690, and 700 mg/kg or at least one ofthese amounts. Dosage levels may include a range of 0.5-700 mg/kg, 1-700mg/kg, 10-700 mg/kg, 25-700 mg/kg, 50-700 mg/kg, 75-700 mg/kg, 100-700mg/kg, 125-700 mg/kg, 150-700 mg/kg, 175-700 mg/kg, 200-700 mg/kg,225-700 mg/kg, 250-700 mg/kg, 275-700 mg/kg, 300-700 mg/kg, 325-700mg/kg, 350-700 mg/kg, 375-700 mg/kg, 400-700 mg/kg, 425-700 mg/kg,450-700 mg/kg, 475-700 mg/kg, 500-700 mg/kg, 525-700 mg/kg, 550-700mg/kg, 575-700 mg/kg, 600-700 mg/kg, 625-700 mg/kg, 650-700 mg/kg,675-700 mg/kg, 1-600 mg/kg, 1-500 mg/kg, 1-400 mg/kg, 1-300 mg/kg, 1-200mg/kg, 1-100 mg/kg, 10-700 mg/kg, 10-600 mg/kg, 10-500 mg/kg, 10-400mg/kg, 10-300 mg/kg, 10-200 mg/kg, 10-100 mg/kg, 20-700 mg/kg, 20-600mg/kg, 20-500 mg/kg, 20-400 mg/kg, 20-300 mg/kg, 20-200 mg/kg, 20-150mg/kg, 20-100 mg/kg, 20-75 mg/kg, 20-50 mg/kg, 30-700 mg/kg, 30-60mg/kg, 30-500 mg/kg, 30-400 mg/kg, 30-300 mg/kg, 30-200 mg/kg, 30-100mg/kg, 40-700 mg/kg, 40-600 mg/kg, 40-500 mg/kg, 40-400 mg/kg, 40-300mg/kg, 40-200 mg/kg, 40-100 mg/kg, 50-700 mg/kg, 50-600 mg/kg, 50-500mg/kg, 50-400 mg/kg, 50-300 mg/kg, 50-200 mg/kg, 50-100 mg/kg, 60-700mg/kg, 60-600 mg/kg, 60-500 mg/kg, 60-400 mg/kg, 60-300 mg/kg, 60-200mg/kg, 60-100 mg/kg, 70-700 mg/kg, 70-600 mg/kg, 70-500 mg/kg, 70-400mg/kg, 70-300 mg/kg, 70-200 mg/kg, 70-100 mg/kg, 80-700 mg/kg, 80-600mg/kg, 80-500 mg/kg, 80-400 mg/kg, 80-300 mg/kg, 80-200 mg/kg, 80-100mg/kg, 90-700 mg/kg, 90-600 mg/kg, 90-500 mg/kg, 90-400 mg/kg, 90-300mg/kg, 90-200 mg/kg, 90-100 mg/kg, 100-700 mg/kg, 100-600 mg/kg, 100-500mg/kg, 100-400 mg/kg, 100-300 mg/kg, 100-200 mg/kg, 200-700 mg/kg,200-600 mg/kg, 200-500 mg/kg, 200-400 mg/kg, 200-300 mg/kg, 300-700mg/kg, 300-600 mg/kg, 300-500 mg/kg, 300-400 mg/kg, 400-700 mg/kg,400-600 mg/kg, 400-500 mg/kg, 500-700 mg/kg, 500-600 mg/kg, 600-700mg/kg, or any range therebetween.

Multiple doses of one or a combination of drugs may be delivered to theindividual periodically, such as one or more times a day, one or moretimes a week, or one or more times a month, for example. The deliverymay be oral, intravenous, subcutaneous, for example, or by other means.The compound of the invention may be delivered as a solid or as a liquidand may require the individual to ingest the compound with food orshortly after ingesting food.

In embodiments of the invention, an individual is diagnosed with sicklecell disease prior to receiving the inventive treatment. Sickle cellanemia may be indicated when abnormal sickle-shaped cells in the bloodare identified, such as microscopically. Testing may alternatively oradditionally include examining a smear of blood using a speciallow-oxygen preparation (referred to as a sickle prep). Additional oralternative tests may be utilized, including to detect the abnormalhemoglobin S (such as solubility tests performed on tubes of bloodsolutions) and/or by specifically quantifying the types of hemoglobinpresent using a hemoglobin electrophoresis test (identifies thehemoglobins in the blood by separating them). In some cases there isprenatal diagnosis utilizing amniocentesis or chorionic villus sampling,for example, and the sample obtained is then tested for DNA analysis ofthe fetal cells; infants, adolescents, and/or adults may also bediagnosed with DNA testing for particular mutation(s) that cause sicklecell.

In embodiments of the invention, one or more compounds are delivered toan individual that reduce one or more side effects of a drug employedfor SCD. In some embodiments, one or more compounds are delivered to anindividual that reduce one or more side effects of HU for the treatmentof any medical condition, including SCD. The one or more compounds mayor may not be inducers of HbF production, in some embodiments. Inspecific cases, for example, the one or more compounds (such ashydroxyfasudil, for example) reduces coagulation and/or reducesinflammation in an individual, including an individual that has been oris being or will be administered HU. Such biological effects byhydroxyfasudil, for example, to improve deleterious side effects of HU,for example, may be direct or indirect.

In certain embodiments of the invention, Isocoronarin D is used alone orwith one or more other compounds in the treatment and/or prevention ofSCD. The Isocoronarin D may be utilized in conjunction withhydroxyfasudil, in certain aspects. In particular embodiments,Isocoronarin D is employed with one or more inducers of HbF production.

In some embodiments, there is a method of treating an individual withsickle cell disease, comprising the step of delivering to the individuala therapeutically effective amount of Hydroxyfasudil. In specific cases,the method is further defined as delivering to the individual atherapeutically effective amount of Hydroxyfasudil and an inducer offetal hemoglobin (HbF) production. In specific embodiments, the inducerof HbF production is selected from the group consisting of hydroxyurea,isocoronarin D, 5-azacytidine, citarabine, butyrates, tricostatin,apicidin, scripaid, mithramycin, cisplatin, tallimustine, angelicin,rapamycin, everolimus, resveratrol, 5-methoxypsoralen, lenalidomide,pomalidomide, triple-helix oligodeoxynucleotides, peptide nucleic acids,and a combination thereof

In certain embodiments, Hydroxyfasudil and the inducer of HbF productionare delivered simultaneously or are delivered separately. In caseswherein there is separate delivery of Hydroxyfasudil and the inducer ofHbF production, the separate delivery of Hydroxyfasudil and the inducerof HbF production may be delivered within minutes, hours, days, weeks,or months of each other. In cases where more than one compound isdelivered to an individual, the delivery of the two compounds may be inthe same way or in different ways.

In specific embodiments of the methods, they further comprise the stepof determining that the individual has sickle cell disease and/ordetermining that a biological family member has sickle cell disease.

In some embodiments of the invention, there are methods of treating anindividual with sickle cell disease, comprising the step of deliveringto the individual a therapeutically effective amount of Isocoronarin D.The methods may be further defined as delivering to the individual atherapeutically effective amount of Isocoronarin D and an inducer offetal hemoglobin (HbF) production. In some embodiments, the methodsfurther comprise delivering another sickle cell disease treatment to theindividual.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description. It is to be expressly understood,however, that the disclosure is provided for the purpose of illustrationand description only and is not intended as a definition of the limitsof the present invention.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

In keeping with long-standing patent law convention, the words “a” and“an” when used in the present specification in concert with the wordcomprising, including the claims, denote “one or more.” Some embodimentsof the invention may consist of or consist essentially of one or moreelements, method steps, and/or methods of the invention. It iscontemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein.

The term “inducer of fetal hemoglobin (or HbF) production” as usedherein refers to one or more compounds that directly or indirectlyincrease the level of fetal hemoglobin or one or more subunits thereof,in a mammalian cell, including increasing the transcription ortranslation of HbF.

The term “therapeutically effective amount” as used herein refers tothat amount which, when administered to a subject or patient fortreating a disease, is sufficient to effect such treatment for thedisease, including to ameliorate at least one symptom of the disease.

II. Certain Embodiments of the Invention

Embodiments of the invention include utilizing Hydroxyfasudil fortreatment of SCD, and in specific embodiments Hydroxyfasudil is utilizedwith one or more other drug compounds. In particular casesHydroxyfasudil is utilized with HU or with another inducer of HbFproduction.

Currently, Hydroxyurea (HU) is the only FDA approved drug capable ofmodifying the SCD processes (Brawley, et al 2008). HU positivelycounteracts the pathophysiology of SCD by increasing the production offetal hemoglobin (HbF)-containing erythrocytes via stimulation of theNO-cyclic guanosine monophosphate (cGMP) signaling pathway (Cokic, et al2003) and indirectly altering gene expression and proteins associatedwith the pathophysiology of SCD. The increased concentration ofHbF-containing erythrocytes dilutes the concentration of sicklederythrocytes thereby sequentially triggering decreased hemolysis (Olnes,et al 2009), increased NO bioavailability (Conran, et al 2004) anddecreased endothelium activation (Haynes, et al 2008). However, HU hasbeen demonstrated to reduce leukocyte counts in patients on therapy(Charache, et al 1996).

It has also been demonstrated that HU therapy induces side effect suchas inducing DNA damage (Friedrisch, et al 2008), reducing sperm counts(Grigg 2007) and producing iron nitrosyl Hb (Lockamy, et al 2003). It isimportant to note that HU increases the expression of pro-inflammatorymolecules such as IL-6 and IL-8 which contributes to the chronicinflammatory state associated with the pathophysiology of SCD (Laurance,et al 2010). Moreover, HU has no demonstrable effects on the levels ofthe pro-coagulatory molecule fibronectin that contributes tovaso-occlusion (Saleh, et al 1999). Furthermore, not all SCD patientsrespond to HU therapy. Therefore, because of the side effects of HUthere is a need for less toxic drugs for the treatment of SCD.

In embodiments of the invention, Hydroxyfasudil is combined with one ormore inducers of HbF production, and in at least some casesHydroxyfasudil alone or in combination with others renders a less toxicand more effective treatment for SCD. Such a combination of compounds(which may be synergistic) is beneficial compared to HU treatmentbecause of advantages such as increased HbF production, reducedinflammation and reduced vaso-occlusion, for example.

Although Hydroxyfasudil has demonstrable effects for treating acuteischemic stroke (Shibuya, et al 2005), stable angina (Vicari, et al2005), cerebral vasospasm (Suzuki, et al 2008), pulmonary hypertension(Doe, et al 2009) and Alzheimer's disease (Huentelman, et al 2009), ithas not yet been experimentally proven that Hydroxyfasudil increases HbFproduction that at least in some cases is useful for SCD therapy andthat effect of HU therapy. Useful effects of the inventive use ofcombination of drugs (in the concentration necessary to bring about thedesired beneficial effects in the treated patient) includes thebeneficial and distinguishing effects (compared to HU) of relievingvaso-occlusive crisis and/or reducing inflammation.

In specific embodiments of the invention, the required HbF productioncan be induced by HbF inducers that are less toxic than HU compared tothe situation when HU is used alone. To this effect, in some embodimentsHU may be used together with Hydroxyfasudil including, for example, indosages smaller than currently administered. In some embodiments,hydroxyfasudil compensates for known HbF inducers' inability toeffectively modulate vaso-occlusion and inflammation associated with theeffective treatment of SCD.

Other embodiments of the invention include a Literature Based Discovery(LBD) approach to identify one or more candidate drugs for the treatmentof SCD that are more suitable treatment than the currently administeredHU therapy.

Exemplary methods are as follows:

1. The inventors used Dragon Knowledge Explorer system to create DESSCD,an LBD tool focused on published scientific literature on SCD and blooddiseases. DESSCD allowed generation of hypotheses based on whichinformation was extracted on blood disease drugs (BDDs) linked tospecific genes and proteins implicated in the pathophysiology of SCD. Intotal, 232,634 SCD-related Medline abstracts were processed.

2. The inventors collated the BDDs that had been linked to all theselected SCD-related molecules and proteins.

3. The inventors extracted the BDDs that have not been screened forapplication as a SCD drug.

4. The inventors hand curated and extracted the BDDs in Step 3 thatinduced the desired effect on the molecules and proteins implicated inthe pathophysiology of SCD.

III. Inducers of HbF Production

One or more inducers of HbF production may be employed in the invention.In specific aspects, the inducer lacks deleterious side effectsassociated with HU treatment. Exemplary inducers of HbF productioninclude hydroxyurea, isocoronarin D, 5-azacytidine, citarabine,butyrates, tricostatin, apicidin, scriptaid, mithramycin, cisplatin,tallimustine, angelicin, rapamycin, everolimus, resveratrol,5-methoxypsoralen, lenalidomide, pomalidomide, triple-helixoligodeoxynucleotides, peptide nucleic acids, or a combination thereof(see Gambari and Fibach, 2007; Moutouh-de Parseval et al., 2008; orBianchi et al., 2007, for example).

Furthermore, in vitro model systems for screening potential inducers offetal hemoglobin are known in the art. For examples, cells transfectedwith reporter genes under the control of ^(G)γ-globin gene promoter maybe employed (see Skarpidi et al., 2000, for example). In other cases,one can utilize reporter genes within an intact β-globin gene locusunder the ^(G)γ-globin promoter (see Vadolas et al., 2004, for example).

IV. Combination Therapies

In some embodiments, one or more compounds useful for treating sicklecell disease or enhancing treatment of another sickle cell disease drugcompound are employed in the invention. In specific embodiments,hydroxyfasudil is employed with at least one other compound in thetreatment of sickle cell disease, and the two or more compounds may actadditively or synergistically.

The administration of the two or more SCD drug compounds may besimultaneous or it may be separate. In such instances where it isseparate, it is contemplated that one may provide an individual withboth compounds within seconds or minutes or days or weeks or months fromeach other. In some cases, one may provide an individual within about1-60 minutes of each other or within 1-24 h of each other or withinabout 6-12 h of each other or within 12-24 h of each other, or within1-7 days of each other, or within 1-4 weeks of each other or 1-12 monthsof each other, for example. In some situations, it may be desirable toextend the time period for treatment significantly, however, whereseveral d (2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8)lapse between the respective administrations.

In some cases, there is substantially simultaneous delivery of themultiple compounds. In certain cases, the two or more SCD compounds aredelivered in a same route, although in specific cases the two or moreSCD compounds are delivered differently (for example, one oral and oneby i.v.).

Administration of the therapeutic compounds of the present invention toa patient will follow general protocols for the administration of drugs,taking into account the toxicity, if any, of the drug. It is expectedthat the treatment cycles would be repeated as necessary. It also iscontemplated that various standard therapies for symptoms of sickle celldisease may be applied in combination with hydroxyfasudil and/or othermedical treatments (such as pain relievers, antibiotics, oxygen therapy,blood transfusions, and so forth).

V. Pharmaceutical Preparations

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more sickle cell disease drug compoundsdissolved or dispersed in a pharmaceutically acceptable carrier. Inparticular embodiments, the pharmaceutical preparation comprises one ormore sickle cell disease drugs, including one or more inducers of HbFproduction. In specific aspects, the kit comprises Hydroxyfasudil and,optionally, another compound including, for example, an inducer of HbFproduction. In some embodiments, the pharmaceutical preparationcomprises Isocoronarin D.

The phrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, suchas, for example, a human, as appropriate. The preparation of apharmaceutical composition that contains at least one compound oradditional active ingredient will be known to those of skill in the artin light of the present disclosure, as exemplified by Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,incorporated herein by reference. Moreover, for animal (e.g., human)administration, it will be understood that preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

The compound may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. The present invention can be administered intravenously,intradermally, transdermally, intrathecally, intraarterially,intraperitoneally, intranasally, intravaginally, intrarectally,topically, intramuscularly, subcutaneously, mucosally, orally,topically, locally, inhalation (e.g., aerosol inhalation), injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in cremes, in lipid compositions(e.g., liposomes), or by other method or any combination of the forgoingas would be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference).

The compound may be formulated into a composition in a free base,neutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as formulated for parenteraladministrations such as injectable solutions, or aerosols for deliveryto the lungs, or formulated for alimentary administrations such as drugrelease capsules and the like.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a the composition contained therein, itsuse in administrable composition for use in practicing the methods ofthe present invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include a sickle celldisease drug compound(s), one or more lipids, and an aqueous solvent. Asused herein, the term “lipid” will be defined to include any of a broadrange of substances that is characteristically insoluble in water andextractable with an organic solvent. This broad class of compounds arewell known to those of skill in the art, and as the term “lipid” is usedherein, it is not limited to any particular structure. Examples includecompounds which contain long-chain aliphatic hydrocarbons and theirderivatives. A lipid may be naturally occurring or synthetic (i.e.,designed or produced by man). However, a lipid is usually a biologicalsubstance. Biological lipids are well known in the art, and include forexample, neutral fats, phospholipids, phosphoglycerides, steroids,terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides,lipids with ether and ester-linked fatty acids and polymerizable lipids,and combinations thereof. Of course, compounds other than thosespecifically described herein that are understood by one of skill in theart as lipids are also encompassed by the compositions and methods ofthe present invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the compound may be dispersed in a solutioncontaining a lipid, dissolved with a lipid, emulsified with a lipid,mixed with a lipid, combined with a lipid, covalently bonded to a lipid,contained as a suspension in a lipid, contained or complexed with amicelle or liposome, or otherwise associated with a lipid or lipidstructure by any means known to those of ordinary skill in the art. Thedispersion may or may not result in the formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

A. Alimentary Compositions and Formulations

In preferred embodiments of the present invention, the compound isformulated to be administered via an alimentary route. Alimentary routesinclude all possible routes of administration in which the compositionis in direct contact with the alimentary tract. Specifically, thepharmaceutical compositions disclosed herein may be administered orally,buccally, rectally, or sublingually. As such, these compositions may beformulated with an inert diluent or with an assimilable edible carrier,or they may be enclosed in hard- or soft-shell gelatin capsule, or theymay be compressed into tablets, or they may be incorporated directlywith the food of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515;5,580,579 and 5,792, 451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, such as, for example,gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof; a disintegrating agent, such as, forexample, corn starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier. Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup of elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

Additional formulations which are suitable for other modes of alimentaryadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum. After insertion, suppositories soften, melt or dissolvein the cavity fluids. In general, for suppositories, traditionalcarriers may include, for example, polyalkylene glycols, triglyceridesor combinations thereof. In certain embodiments, suppositories may beformed from mixtures containing, for example, the active ingredient inthe range of about 0.5% to about 10%, and preferably about 1% to about2%.

B. Parenteral Compositions and Formulations

In further embodiments, the sickle cell disease drug compound may beadministered via a parenteral route. As used herein, the term“parenteral” includes routes that bypass the alimentary tract.Specifically, the pharmaceutical compositions disclosed herein may beadministered for example, but not limited to intravenously,intradermally, intramuscularly, intraarterially, intrathecally,subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308,5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specificallyincorporated herein by reference in its entirety).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in isotonic NaCl solution andeither added hypodermoclysis fluid or injected at the proposed site ofinfusion, (see for example, “Remington's Pharmaceutical Sciences” 15thEdition, pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

C. Miscellaneous Pharmaceutical Compositions and Formulations

In other preferred embodiments of the invention, the active compound maybe formulated for administration via various miscellaneous routes, forexample, topical (i.e., transdermal) administration, mucosaladministration (intranasal, vaginal, etc.) and/or inhalation.

Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-solubly based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andluarocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch”. For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays has been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

VI. Kits of the Invention

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, a sickle cell drug compound (s) may be comprisedin a kit. In particular embodiments, the kit comprises one or moresickle cell drug compounds, including one or more inducers of HbFproduction. In specific aspects, the kit comprises hydroxyfasudil and,optionally, another compound including, for example, an inducer of HbFproduction. In some embodiments, the kit comprises isocoronarin D.

The kits may comprise a suitably aliquoted sickle cell drug compound,lipid and/or additional agent compositions of the present invention. Thecomponents of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there is more than one component in the kit, the kitalso will generally contain a second, third or other additionalcontainer into which the additional components may be separately placed.However, various combinations of components may be comprised in a vial.The kits of the present invention also will typically include a meansfor containing the sickle cell drug compound(s) and any other reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow molded plastic containers into which thedesired vials are retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The compositions may alsobe formulated into a syringeable composition, in which case, thecontainer means may itself be a syringe, pipette, and/or other such likeapparatus, from which the formulation may be applied to an infected areaof the body, injected into an animal, and/or even applied to and/ormixed with the other components of the kit. However, the components ofthe kit may be provided as dried powder(s). When reagents and/orcomponents are provided as a dry powder, the powder can be reconstitutedby the addition of a suitable solvent. It is envisioned that the solventmay also be provided in another container means.

Irrespective of the number and/or type of containers, the kits of theinvention may also comprise, and/or be packaged with, an instrument forassisting with the injection/administration and/or placement of theultimate sickle cell drug compound(s) within the body of an animal. Suchan instrument may be a syringe, pipette, forceps, and/or any suchmedically approved delivery vehicle, for example.

EXAMPLES

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way,however, be construed as limiting the broad scope of the invention.

Example 1 Exemplary Identification of SCD Drugs

Current research focuses on disease modifying drugs and curativestrategies such as stem cell transplantation (Bernaudin, et al 2007),gene therapy (Sadelain 2006) and Haemoglobin F (HbF)-inducers (Ley, etal 1983), as these are likely to have the most impact on SCD patients.Nonetheless, the morbidity and sequelae of the disease remains high.Efforts toward discovery of disease modifying drugs can be augmented byleveraging the plethora of molecular data in published biomedicalliterature, as available chemicals with pharmaceutical effect associatedwith an erythrocyte disease other than SCD may be more suitablecandidate for the treatment of SCD than HU. In this endeavor, as of Feb.28, 2011, 232,634 SCD-related MEDLINE abstracts were retrieved fromPubMed, 34.64% (80,574) of which were published in the last decade. Thisvolume of biomedical data cannot be processed by a single researcher orresearch group in a reasonable time. Thus, the inventors haveimplemented a text-mining approach that allows for the summarization ofthis large volume of raw data by automatically distilling theinformation, extract text, discovering implicit links by association andgenerating hypotheses. We used a literature based discovery (LBD) toolDES (Dragon Exploration System) to generate hypotheses leading to theidentification of chemicals with pharmaceutical effect associated withan erythrocyte disease other than SCD may be more suitable candidate forthe treatment of SCD than HU. This methodology allowed for theidentification of Hydroxyfasudil as a SCD drug, in certain embodiments.The inventors additionally hand curated the PubMed literature toidentify potential SCD drugs that may have been omitted as themethodology only allows for the inclusion of well researched potentialSCD drugs.

The Study allowed for the identification of Hydroxyfasudil or Fusadil(5-(1,4-diazepane-1-sulfonyl)isoquinolin-1-ol), a recognized Rho-kinaseinhibitor as a potential novel SCD drug. A comparative analysis ofHydroxyurea and Hydroxyfasudil in relation to SCD displays theadvantages of using Hydroxyfasudil to treat SCD (Table 6).

TABLE 6 Effects by Hydroxyfasudil (HF) compared to HU related treatmentfor SCD Expression status in Molecules Type of molecule SCD ReferencesEffect of HU Effect of HF ROS IR injury-related up (Huang, et al notknown decrease molecule 2007) NO vasodilator down (Eberhardt, etincrease increase al 2003) ET-1 vasoconstrictor up (Werdehoff, decreasedecrease et al 1998) ET-3 vasoconstrictor up (Makis, et al decrease notknown 2004) NFkB Activator of endothelial up (Belcher, et al decreasedecrease cells 2005) HIF-1 Activator of endothelial up (Kim, et al notknown decrease cells 2006) TF Activator of endothelial up (Solovey, etnot known decrease cells al 1998) eNOS Inhibitor of endothelial down(Solovey, et increase increase cell activation al 2010) TNFαcytokine/inflammatory up (Lanaro, et al decrease decrease 2009) IL-1βcytokine/inflammatory up (Wanderer increase decrease 2009) IL-8chemokine/inflammatory up (Lanaro, et al increase not known 2009) IL-6chemokine/inflammatory up (Croizat increase decrease 1994) IL-3inflammatory up (Croizat increase not known 1994) GM-CSF inflammatory up(Conran, et al decrease not known 2007) IL-10 Anti-inflammatory down(Lanaro et al. increase increase 2009) VCAM-1 adhesion up (Solovey, etdecrease not known al 1997) ICAM-1 adhesion up (Solovey, et decreasedecrease al 1997) SELP adhesion up (Solovey, et decrease decrease al1997) SELE adhesion up (Solovey, et decrease decrease al 1997) VLA-4adhesion up (Gambero, et decrease not known al 2007) CD36 adhesion up(Gambero, et decrease not known al 2007) FN1 coagulatory up (Saleh et noeffect decrease al.1999) increase = increases protein levels decrease =reduces protein levels no effect = exert no effect on protein levels notknown = has not been experimentally determined

The inventors additionally hand curated the literature to identifypotential drugs for the treatment of SCD. Isocoronarin D (Chokchaisiri,et al 2010) was identified as a useful drug in certain embodiments forthe treatment of SCD as experimental evidence demonstrates its abilityto induce the production of fetal HbF. (see Example 2)

Comparative analysis of HU and Hydroxyfasudil in relation to SCDindicates that Hydroxyfasudil should be combined with inducer(s) of HbFin order to combat SCD more efficiently. Hydroxyfasudil has severaldistinctive features that HU does not possess that results in reducedvaso-occlusion and reduced inflammation.

Example 2 Isocoronarin D Embodiments

As mentioned above, the effectiveness of HU for the treatment of SCD isa consequence of its ability to produce HbF via stimulation of theNO-cyclic guanosine monophosphate (cGMP) signaling pathway (Cokic, et al2003). Isocoronarin D induces the production of HbF, however, it alsodisplays the propensity to induce HbF production with little to noassociated toxicity.

A PubMed search using keywords “fetal hemoglobin production AND drug NOTsickle cell” retrieved 84 Medline article from which only Isocoronarin D(Chokchaisiri, et al 2010) was identified as a potential drugs for thetreatment of SCD as experimental evidence demonstrates its ability toinduce the production of fetal HbF that is the primary factor for theeffectiveness of HU therapy for SCD.

Methodology:

1. The inventors used compiled based on Dragon Knowledge ExplorationSystem (DKES) the exploration system for SCD, a Literature BasedDiscovery (LBD) tool focused on literature for SCD and blood diseases togenerate hypotheses that allowed teh inventors to extract blood diseasedrugs (BDDs) that have demonstrated the ability to induce HbFproduction.

2. The inventors hand curated the BDDs that had been linked to HbFproduction but have not been screened for application as SCD drugs.

The study allowed for the identification if Isocoronarin D. IsocoronarinD was isolated for the first time from the rhizome of Hedychiumcoronarium (Zingiberaceae), collected in the Kathmandu Valley, NepalSingh et al., 1991). A study by Chockchaisiri et al. demonstrates HbFproduction using a K562 reporter cell line harboring the enhanced greenfluorescence protein (EGFP) gene under the control of a (G) gamma-globinpromoter. In this assay, the reporter cell lines were treated with theknown HbF inducers, hemin and cisplatin, and seven diterpenes. Of theseven diterpenes, Isocoronarin D exhibited the highest EGFP inducingpotency or HbF production (Chokchaisiri, et al 2010). They alsoevaluated the degree of cytoxicity associated with the IsocoronarinD-induced HbF production and showed that at optimal concentration forHbF production, Isocoronarin D maintains cell viability of 75-80%(Chockchaisiri et al 2010).

REFERENCES

All patents and publications mentioned in this specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications herein are incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by referencein their entirety.

Patents and Patent Applications

-   U.S. Pat. No. 5,399,363-   U.S. Pat. No. 5,466,468-   U.S. Pat. No. 5,543,158-   U.S. Pat. No. 5,580,579-   U.S. Pat. No. 5,641,515-   U.S. Pat. No. 5,725,871-   U.S. Pat. No. 5,756,353-   U.S. Pat. No. 5,780,045-   U.S. Pat. No. 5,792,451-   U.S. Pat. No. 5,804,212-   U.S. Pat. No. 6,613,308-   U.S. Pat. No. 6,7537,514

Publications

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(2006) Heme oxygenase-1 is a    modulator of inflammation and vaso-occlusion in transgenic sickle    mice. J Clin Invest, 116, 808-816.-   Belcher, J. D., Mahaseth, H., Welch, T. E., Vilback, A. E.,    Sonbol, K. M., Kalambur, V. S., Bowlin, P. R., Bischof, J. C.,    Hebbel, R. P. & Vercellotti, G. M. (2005) Critical role of    endothelial cell activation in hypoxia-induced vasoocclusion in    transgenic sickle mice. Am J Physiol Heart Circ Physiol, 288,    H2715-2725.-   Bernaudin, F., Socie, G., Kuentz, M., Chevret, S., Duval, M.,    Bertrand, Y., Vannier, J. P., Yakouben, K., Thuret, I., Bordigoni,    P., Fischer, A., Lutz, P., Stephan, J. L., Dhedin, N., Plouvier, E.,    Margueritte, G., Bones, D., Verlhac, S., Esperou, H., Coic, L.,    Vernant, J. P. & Gluckman, E. (2007) Long-term results of related    myeloablative stem-cell transplantation to cure sickle cell disease.    Blood, 110, 2749-2756.-   Bianchi, N., Zuccato, C., Lampronti, I., Borgatti, M., and    Gambari, R. 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(2006) The synergy site of    fibronectin is required for strong interaction with the platelet    integrin alphaIIbbeta3. Ann Biomed Eng, 34, 1542-1552.-   Charache, S., Barton, F. B., Moore, R. D., Terrin, M. L.,    Steinberg, M. H., Dover, G. J., Ballas, S. K., McMahon, R. P.,    Castro, 0. & Orringer, E. P. (1996) Hydroxyurea and sickle cell    anemia. Clinical utility of a myelosuppressive “switching” agent.    The Multicenter Study of Hydroxyurea in Sickle Cell Anemia. Medicine    (Baltimore), 75, 300-326.-   Chiu, J. J., Lee, P. L., Chen, C. N., Lee, C. I., Chang, S. F.,    Chen, L. J., Lien, S. C., Ko, Y. C., Usami, S. & Chien, S. (2004)    Shear stress increases ICAM-1 and decreases VCAM-1 and E-selectin    expressions induced by tumor necrosis factor-[alpha] in endothelial    cells. Arterioscler Thromb Vasc Biol, 24, 73-79.-   Chokchaisiri, R., Chaneiam, N., Svasti, S., Fucharoen, S.,    Vadolas, J. & Suksamrarn, A. 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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

What is claimed is:
 1. A method of treating an individual with sicklecell disease, comprising the step of delivering to the individual atherapeutically effective amount of Isocoronarin D.
 2. The method ofclaim 1, further defined as delivering to the individual atherapeutically effective amount of Isocoronarin D and an inducer offetal hemoglobin (HbF) production.
 3. The method of claim 1, furthercomprising delivering another sickle cell disease treatment to theindividual.